The long-range objective of this research proposal is to develop a thorough understanding of the functional activities of gamma secretase at the atomic level. Gamma Secretase is a membrane protease complex that cleaves the C-terminal end of the amyloid precursor protein (APP). This processing of APP together with its cleavage at the N-terminal end by ?-secretase produces amyloid ? fragments (A??), which form amyloid plaques that are characteristically present in the brain of Alzheimer's patients. [unreadable] Presenilin-1, a component of gamma secretase, contains the enzymatic active site. Mutations of presenilin-1 have been associated with the cause of familial Alzheimer's disease; presenilin-1 is therefore a potential target for therapeutic drugs. The atomic structure of gamma secretase can be expected to provide us with the molecular details of this novel enzyme complex. This structural information together with those of its mutant forms can be expected to yield insights into the mechanism of its proteolytic activity within the membrane bilayer and the change in the proteolytic pattern found in mutant forms of gamma secretase that are linked to Alzheimer's disease. Such structural information will help in the design of therapeutic drugs. The specific aim of this proposal is to purify and crystallize gamma secretase complex in order to obtain crystals suitable for subsequent crystallographic structure determination. Initially, we will continue to develop the purification of gamma secretase from HeLa cells and will perform 3D crystallization trials utilizing a recently developed microfluidic technology or 2D crystallization trials. Alternatively, we will overexpress gamma secretase using a baculovirus overexpression system in order to obtain milligram quantities of the protein complex for "conventional" 3D crystallization trails. The baculovirus overexpression system will also allow us to produce mutant proteins for biochemical and structural studies. [unreadable] [unreadable]